Systems that acquire images of luminescently-labeled microspheres have proven useful for assays. In some such systems, images of assayed microspheres are acquired at multiple emission wavelengths. The luminescent spectral code and fluorescence from each microsphere can be read by superimposing the multispectral images of the same Field of View (FOV) after the application of an image segmentation algorithm. However, in this scheme, microspheres in each of the multispectral images need to be localized separately before the superimposition. The analysis is computationally intensive and the processing time increases proportionally with the increase in number of colors (i.e., wavelengths).
Another method involves localizing the positions of microspheres in an array using forward scatter measurements and then reading the luminescence at multiple wavelengths from those locations (Kamentsky et al, “Micro-Based Multiparameter Laser Scanning Cytometer Yielding Data Comparable to Flow Cytometry Data,” Cytometry 12, 381-387 (1991); and U.S. Pat. Nos. 5,885,840 and 5,072,382). Several variations of this method have been proposed (see, for example, U.S. Pat. Nos. 6,970,246, 6,759,235, 6,656,683 and RE38,817). However, in all of these methods, the imaging apparatus requires a different sensor (such as a photomultiplier tube) to measure the forward scatter signal. While sensors measuring luminescence may be present on the same side of the specimen as the excitation source, the sensor measuring forward scatter is positioned on the opposite side of the specimen such as in Laser Scanning Cytometer (Compucyte, Inc.) (see, for example, Kamentsky et al., “Micro-Based Multiparameter Laser Scanning Cytometer Yielding Data Comparable to Flow Cytometry Data,” Cytomety 12, 381-387 (1991); and U.S. Pat. No. 5,072,382). An imaging system with multiple sensors to image the same FOV is more susceptible to optical alignment problems and prone to errors in the signal measurements.